KR100739232B1 - Pleuropterus ciliinervis extracts showing apoptosis inhibitive effect - Google Patents

Abstract

The present invention relates to nadohasuo extract exhibiting apoptosis inhibitory activity, and more particularly, by showing that nadohasuo extract or ememodine has apoptosis inhibitory activity, nadohasuo extract, ememodine or a pharmaceutically acceptable salt thereof. Pharmaceutical composition containing as an active ingredient can be used for the prevention and treatment of inflammatory diseases caused by increased apoptosis, acquired immunodeficiency syndrome, neurodegenerative diseases, ischemic diseases including stroke and liver diseases caused by toxic substances such as alcohol. To a red composition.

Pleuropterus ciliinervis, apoptosis, emodin

Description

Pleuropterus ciliinervis extracts showing apoptosis inhibitive effect

The present invention relates to nadohasuo extract exhibiting apoptosis inhibitory activity, and more particularly, by showing that nadohasuo extract or ememodine has apoptosis inhibitory activity, nadohasuo extract, ememodine or a pharmaceutically acceptable salt thereof. Pharmaceutical composition containing as an active ingredient can be used for the prevention and treatment of inflammatory diseases caused by increased apoptosis, acquired immunodeficiency syndrome, neurodegenerative diseases, ischemic diseases including stroke and liver diseases caused by toxic substances such as alcohol. To a red composition.

Apoptosis or programmed cell death (PCD) is the basic intracellular process for spontaneous self-elimination of damaged or unwanted cells in a wide range of biological systems. Apoptosis is an essential mechanism for organ development, tissue remodeling, maintenance of intracellular homeostasis and removal of abnormal and damaged cells because the contents of cells that have died through the process of apoptosis are not extracellularly released and do not damage other cells. .

As mentioned above, the apoptosis process, which plays an important role in living organisms, must be precisely regulated according to a predetermined program. If apoptosis is inhibited, diseases such as cancer, autoimmune disease and viral infectious diseases are induced. Kerr et al., Br. J. Cancer, 26, 239-257, 1972], Inappropriately increasing apoptosis leads to diseases such as acquired immunodeficiency, neurodegenerative diseases, ischemic diseases including stroke, and liver diseases caused by toxic substances such as alcohol.

Early cells undergoing apoptosis develop chromatin aggregation, cytoplasm, and the like, which are induced during late stages of damage such as cell junctions, blebbing of cell membranes, and cell shrinkage. Nuclear enrichment (cytoplasm and nuclei condensation), loss of mitochondrial membrane potential, changes in plasma membrane composition, and apoptotic body formation are induced to undergo morphological and biochemical changes throughout. DNA fragmentation in the form of oligonucleosomes, the final result of apoptosis, is a typical biochemical feature of cells undergoing apoptosis [Green DR, and Reed, JC, Science, 281, 1309-1312, 1998].

Many apoptosis-related studies have now identified key players and mechanisms involved in apoptosis in a variety of organisms. Representatively, in mammalian cells, caspase, a type of cellular cysteine protease, is known to play an important role in regulating apoptosis mechanism, which is estimated based on the homology of amino acid sequence. According to the evolutionary phylogenetic tree can be divided into three types centered on ICE (interlukin 1 converting enzyme), CPP32, ICH-1.

Caspases may be receptor-mediated signal transduction, depletion of growth factors, oxidative stress, DNA damage and cell-cells. ) Or activated by a variety of stimuli, including disruption of cell-matrix interactions, poly (ADP-ribose) polymerase (RARP), lamine, Induces apoptosis formation by breaking down cellular proteins such as cytokeratins and caspase-activated DNase inhibitors (ICADs) [Cryns, V., and Yuan, J., Genes Dev., 12, 1551-1570, 1998].

Active studies of apoptosis have shown that in mammals caspases belonging to at least 14 different classes so far, all of which are peptide substrates after aspartic acid residues, which play a pivotal role in the initiation and execution of apoptosis. Has been reported to induce apoptosis by cleaving (Nunez et al., Oncogene, 17, 3237-3245, 1998).

Substances with apoptosis inducing activity have been reported that can be used to induce cell death in various cancers where uncontrolled growth and proliferation of abnormal cells occurs [Siegel, D.S. et al., Proc. Natl. Acad. Sci. 6; 95 (1), 162-6, 1998].

In contrast, compounds with such apoptosis inhibitory activity are inflammatory diseases, especially inflammatory diseases caused by increased apoptosis [Lee et al., BBRC, 302, 539-544, 2003], acquired immunodeficiency [Donnerer J., Pharmacology, 269, 197-204, 2003], neurodegenerative diseases such as Alzheimer's disease and Parkinson's disease [Li et al., FEBS Lett., 21, 261-269, 2004], ischemic diseases including stroke [Gotz et al., J. Neurosci . Res., 56, 420-426, 1999], and it has been reported that it can be used for the treatment and prevention of liver diseases caused by toxic substances such as alcohol [Vanesa et al., Life Sci., 24, 683-692, 2005]. .

Emodine is a compound that has been extracted from rhubarb and the like, and is known as a substance that activates cascade and induces apoptosis [Lee, H.Z., Br. J. Pharmaco., 134 (5), 1093-1103, 2001; Srinivas et al., Eur. J. Pharmaco., 473 (2/3), 117-125, 2003; Shieh et al., Life Sciences, 74 (18), 2279, 2004], have not been reported to inhibit apoptosis.

Therefore, the present inventors searched for a substance having apoptosis inhibitory activity from natural products, and separated the active ingredient from Pleuropterus ciliinervis , and carried out a physicochemical analysis thereof, wherein the active ingredient is represented by the following formula I The present invention was completed by revealing that it is (emodin) and revealing that emodin exhibits apoptotic inhibitory activity that has not been reported before.

Emodine has been reported previously that it can be used to induce cell death in various cancers, but the present invention was first found to have apoptosis inhibitory activity, inflammatory diseases caused by increased apoptosis, acquired immunodeficiency disease, neurodegenerative diseases, stroke Ischemic diseases, including alcohol, for the first time to be used for the treatment and prevention of liver diseases caused by toxic substances.

It is also an object of the present invention to provide a method for separating compounds having apoptosis inhibitory activity from Nado sewage using methanol extraction, ethyl acetate extraction and chromatography.

It is another object of the present invention to provide a pharmaceutical composition for apoptosis inhibitors containing the emodin or the pharmaceutically acceptable salt as an active ingredient.

The present invention is selected from the group consisting of inflammatory diseases, acquired immunodeficiency syndrome, neurodegenerative diseases, ischemic diseases including stroke, and liver diseases caused by toxic substances such as alcohol and inflammatory diseases due to increased apoptosis containing Pleuropterus ciliinervis extract as an active ingredient. It is characterized by a pharmaceutical composition for the prevention or treatment of diseases.

In addition, the present invention is characterized by another method for preparing an extract having apoptosis inhibitory activity from Nado sewage, and separating the active ingredient.

Referring to the present invention in more detail as follows.

The present invention is Nado sewage extract, emodin or pharmaceutically acceptable salts thereof, inflammatory diseases caused by increased apoptosis, acquired immunodeficiency syndrome, neurodegenerative diseases, ischemic diseases including stroke and toxic substances such as alcohol By revealing that it can be usefully used for inhibiting or preventing liver disease, etc., it relates to a pharmaceutical composition containing nadohasuo extract, emodine or a pharmaceutically acceptable salt thereof as an active ingredient.

First, a method for producing a nado sewage extract having apoptosis inhibitory activity from nado sewage is obtained by cutting the nado sewage, cutting it under reflux for 40 to 50 hours with methanol, filtering under reduced pressure, and then extracting with ethyl acetate to prepare the nado sewage extract. do.

In addition, a method of separating the active ingredient having an apoptosis inhibitory activity from Nado sewage is as follows.

1) preparing nado sewage extract as described above;

2) collecting the active fraction by dissolving the Nado sewage extract in methanol and then performing gel filtration chromatography; And

3) The active fraction is separated by high pressure liquid chromatography, the solvent is removed with a reduced pressure dryer, and freeze-dried to obtain an active ingredient of apoptosis inhibitory activity.

In more detail, the nado sewage extract extracted with ethyl acetate is first dissolved in methanol, and then active fractions are collected by performing Sephadex LH-20 gel column chromatography.

The active fraction was then separated by high pressure liquid chromatography using a C18 column and a water-acetonitrile eluting solvent (20% acetonitrile to 100% acetonitrile, 30 minutes). An active ingredient having apoptosis inhibitory activity is obtained from an active fraction of retention time of 33 minutes.

As a result of analyzing the physicochemical properties of the active ingredient obtained by the method such as electron spray ionization mass spectrometer (ESI-MS), nuclear magnetic resonance spectra, it was confirmed that the compound had the following physicochemical properties.

1) Material Property: Powder 2) Molecular Weight: 270

3) Molecular formula: C ₁₅ H ₁₀ O ₅ 4) Mass spectrometry (MH): 269 ( m / z )

In addition, hydrogen nuclear magnetic resonance spectra and carbon nuclear magnetic resonance spectra obtained by using methanol (CD ₃ OD) as a solvent, and the compound was found to be emodin of Formula 1 or 1,3,8- It was found to be a compound known as trihydroxy-2-methyl-anthraquinone.

[Formula 1]

The present invention is characterized by a pharmaceutical composition for apoptosis inhibitors containing Nadohasuo extract, emodin represented by the formula (1) or a pharmaceutically acceptable salt thereof as an active ingredient.

Emodine used in the present invention can be isolated from natural products such as nado sewage, or can be prepared by conventional organic synthesis.

The nado sewage extract or emodine may be useful for inhibiting or preventing inflammatory diseases, acquired immunodeficiency, neurodegenerative diseases, ischemic diseases including stroke, and liver diseases caused by toxic substances such as alcohol, etc. due to an increase in apoptosis. It was confirmed that the apoptosis inhibitory activity is excellent.

On the other hand, the active ingredient of the present invention emodine can be used in the form of a pharmaceutically acceptable salt, the salt is an acid addition salt formed by a pharmaceutically acceptable free acid is useful. Emodines can form pharmaceutically acceptable acid addition salts according to methods conventional in the art. Free acid and inorganic acid may be used as the free acid, and hydrochloric acid, bromic acid, sulfuric acid, phosphoric acid, etc. may be used as the inorganic acid, and citric acid, acetic acid, lactic acid, tartaric acid, maleic acid, and fumaric acid may be used as the organic acid. (fumaric acid), formic acid, propionic acid, oxalic acid, trifluoroacetic acid, benzoic acid, gluconic acid, methanesulfonic acid, glycolic acid, succinic acid, 4-toluenesulfonic acid, galluxuronic acid, embonic acid, glutamic acid or aspartic acid Can be used.

Therefore, the composition containing the nadohasuo extract of the present invention, emodine or a pharmaceutically acceptable salt thereof as an active ingredient is an inflammatory disease due to increased apoptosis, acquired immunodeficiency, neurodegenerative diseases, ischemic diseases including stroke and alcohol, etc. It can be used for the prevention or treatment of liver disease caused by toxic substances.

The compositions of the present invention may be administered orally or parenterally, for example, intravenously, subcutaneously, intraperitoneally or topically, during clinical administration and may be used in the form of general pharmaceutical formulations.

Pharmaceutical compositions of the present invention may be formulated for oral administration, for example tablets, troches, lozenges, aqueous or oily suspensions, prepared powders or granules, emulsions, hard or soft capsules, syrups or elixirs. formulated as elixirs. Binders such as lactose, saccharose, sorbitol, mannitol, starch, amylopectin, cellulose or gelatin for preparation in formulations such as tablets and capsules; Excipients such as dicalcium phosphate; Disintegrants such as corn starch or sweet potato starch; Lubricants such as magnesium stearate, calcium stearate, sodium stearyl fumarate or polyethylene glycol wax. In the case of the capsule formulation, in addition to the substances mentioned above, it contains a liquid carrier such as fatty oil.

In addition, the pharmaceutical composition of the present invention can be administered orally, and oral administration is by subcutaneous injection, intravenous injection, intramuscular injection or intrathoracic injection injection. To formulate into a non-oral dosage form, the compound of formula 1 is mixed in water with a stabilizer or buffer to prepare a solution or suspension, which is formulated in unit dosage forms of ampoules or vials.

The effective dose of emodine represented by Formula 1 is generally 1 to 50 mg / day per kg of adult patient weight, preferably 5 to 20 mg / day, 1 at regular time intervals as determined by the physician or pharmacist. It may be administered several times a day, preferably two to three times a day.

In addition, as a result of acute toxicity test for emodin, MLD (mouse lethal dosage) value was very stable more than 500 mg / kg.

Hereinafter, the present invention will be described in more detail based on the following examples, but the present invention is not limited thereto.

Example 1 Preparation of Nadohasuo Extract

In order to search for compounds capable of modulating apoptosis mechanisms, the inventors prepared extracts from Nadohasuo.

First, 1 kg of nado sewage purchased from Ilshin Chemical was extracted with 100% methanol for 48 hours, concentrated under reduced pressure, and extracted with ethyl acetate to prepare nado sewage extract.

Example 2 Isolation of Apoptosis Inhibitory Activity

In order to search for compounds capable of modulating apoptosis mechanisms, the inventors used extraction and chromatography to separate compounds from Nadohasuoh.

First, the Nado sewage extract prepared as in Example 1 was concentrated, and the active material was collected by dissolving the concentrate in 2 ml of methanol and performing Sephadex LH gel column chromatography. High pressure liquid chromatography (column: C18, flow rate: 1.5 mL / min, 220 nm detection) using the active fraction as an elution solvent for water-acetonitrile (20% acetonitrile to 100% acetonitrile, 30 minutes) An active fraction of 33 minutes holding time was separated, and the solvent was removed by a vacuum dryer, and the residue was lyophilized to obtain 3.0 mg of the compound of Formula 1.

Example 3: Physicochemical Characterization of the Compound Obtained in Example 2

In order to analyze the physicochemical properties of the compound represented by Formula 1 obtained in Example 2, the inventors of the present invention have carried out electrospray ionization mass spectrometry (ESI-MS), Fisons VG Quattro 400 mass spectrometer, USA, and hydrogen and carbon nuclear magnetic resonance spectra. Etc. were used. In the NMR experiment, each sample was dissolved in methanol (CD ₃ OD) solvent and measured in a 5 mm NMR tube. The chemical shift was measured based on the peak of each solvent or the peak of TMS (tetramethylsilane). . As a result of analyzing the property, molecular weight, molecular formula and mass of the compound, the compound of the present invention was found to have the following physical and chemical properties.

As a result, the material property of the compound of Formula 1 was powder, the molecular weight was 270, the molecular formula was confirmed as C ₁₅ H ₁₀ O ₅ , and the mass spectrometry (MH) ⁻ of the compound was 269 ( m / z ). Hydrogen nuclear magnetic resonance spectra and carbon nuclear magnetic resonance spectra of methanol (CD ₃ OD) as a solvent, together with the physicochemical properties, were found to be emodin or 1 according to the present invention. It was found to be a compound known as 3,8-trihydroxy-2-methyl-anthraquinone.

<NMR data>

Compound of Formula 1

¹³ C NMR (CD ₃ OD) δ 21.2, 106.3, 109.6, 119.2, 119.8, 123.1, 123.6, 140.7, 142.2, 142.5, 158.5, 160.0, 162.1, 187.0

Experimental Example 1 Determination of Apoptosis Inhibitory Activity of Nadohasuo Extract and Emodine Isolated therefrom

In order to measure the apoptosis inhibitory effect of Nadohasuo extract and Emodine obtained in Example 1, the present inventors performed the following in vitro experiment using U937 cells, a human blood cancer cell line.

U937 cells were cultured in 96-well microplate until ⁵ × 10 ⁶ cells / well, and then 10 μg / ml of apoptotic inducer etoposide and 1 to 100 μg / ml of the compound of the present invention. apoptosis inhibitory activity was compared to culture for 5 h at 37 ℃ with 95% air condition-concentration process on the U937 cells and 5% CO ₂ in. At this time, cells treated with only etoposide were used as the positive control group, and cells without treatment with both etoposide and the nado sewage extract and ememodine of the present invention were used as the negative control group. After 5 hours, the shape of U937 cells was observed under the microscope to determine apoptosis. ) Was used to measure caspase-3 activity. The compounds of the present invention exhibit the caspase-3 activity of the positive control group (only treated with etoposide) and the caspase-3 activity of the negative control group (untreated with etoposide, nadohasuo extract and emodine). Apoptosis inhibitory effects were determined and shown in Table 1 below.

Comparison of Apoptosis Inhibitory Activity in U937 Cell Lines compound Inhibition rate (IC ₅₀ , Μg / ml) Nadohasuo Extract 65.5 Emodin 10.0 Pyrrolidine dithiocarbonate (PDTC) 15.0

As shown in Table 1, the inhibitory activity concentrations of Nadohasuo extract and emodine (IC ₅₀ , 50% of the inhibitory effect required to show the inhibitory effect) on apoptosis of 937 cells induced by etoposide were 65.5 and 10.0 μg, respectively. / Ml and the inhibitory activity concentration of 15.0 ㎍ / ㎖ of PDTC (pyrrolidine dithiocarbonate) as a standard comparative material, it can be seen that Nado sewage extract and emotein according to the present invention has a superior apoptosis inhibitory effect than PDTC as a standard comparative material there was. From the above results, it was confirmed that the emodine of the present invention is a compound derived from a natural product capable of inhibiting apoptosis in vitro.

Experimental Example 2: Oral Administration of Rats Acute Toxicity Test

Acute toxicity test was performed using 6-week-old SPF SD rats. Two animals per group were suspended orally in a dose of 1 g / kg / 1 ml by emulsification in 0.5% methylcellulose solution each. After administration of the test substance, mortality, clinical symptoms, and changes in body weight were observed. Hematological and hematological examinations were performed. Necropsy was performed to observe abdominal and thoracic organ abnormalities. As a result, there were no clinical symptoms or deaths in all animals treated with the test substance, and no toxicity change was observed in weight change, blood test, blood biochemistry test, autopsy findings, etc. As a result, all of the tested compounds did not show toxic changes up to 500 mg / kg in rats, and the minimum lethal dose (LD ₅₀ ) was determined to be a safe substance of 500 mg / kg or more.

As confirmed above, the emodine of the present invention represented by Formula 1 exhibited an excellent apoptosis inhibitory effect and thus could be prepared as a pharmaceutical composition for apoptosis inhibitors.

Formulation Example 1 Preparation of Syrup

A syrup containing 2% (weight / volume) of an active ingredient of emodine and a pharmaceutically acceptable salt thereof represented by Chemical Formula 1 of the present invention was prepared by the following method.

Acid addition salts of emodin, saccharin and sugars were dissolved in 80 g of warm water. After the solution was cooled, a solution consisting of glycerin, saccharin, spices, ethanol, sorbic acid and distilled water was prepared and mixed. Water was added to this mixture to 100 ml. The addition salt can be replaced with other salts according to the examples.

The components of the syrup are as follows.

Emodine hydrochloride ..... 2 g

Saccharin ......................... 0.8 g

Sugar ............... 25.4 g

Glycerin ......................... 8.0 g

Spice .............. 0.04 g

Ethanol ......................... 4.0 g

Sorbic Acid ............... 0.4 g

Distilled Water .....................

Formulation Example 2: Preparation of Tablet

A tablet containing 15 mg of active ingredient was prepared by the following method.

250 g of emodin-hydrochloride were mixed with 175.9 g of lactose, 180 g of potato starch and 32 g of colloidal silicic acid. 10% gelatin solution was added to the mixture, which was then ground and passed through a 14 mesh sieve. It was dried and tablets were prepared by adding 160 g of potato starch, 50 g of talc and 5 g of magnesium stearate.

Formulation Example 3: Preparation of Injection

Injection solution containing 10 mg of the active ingredient was prepared by the following method.

1 g of emodin-hydrochloride, 0.6 g of sodium chloride and 0.1 g of ascorbic acid were dissolved in distilled water to make 100 ml. The solution was placed in a bottle and heated at 20 ° C. for 30 minutes to sterilize to prepare an injection solution.

As described above, the nado sewage extract and the emodine isolated therefrom according to the present invention have no toxicity and exhibit excellent apoptosis inhibitory effects, and thus, ischemic diseases including inflammatory diseases, acquired immunodeficiency disorders, neurodegenerative diseases, and strokes due to increased apoptosis. It can be usefully used for inhibiting or preventing liver diseases caused by diseases and toxic substances such as alcohol.

Claims (4)

A pharmaceutical composition for preventing or treating liver disease caused by a toxic substance due to an increase in apoptosis containing Pleuropterus ciliinervis extract as an active ingredient. delete delete delete